Purpose Magnetic resonance imaging (MRI) findings in multiple sclerosis (MS) overlap with numerous MS mimics. The central vein sign (CVS) can help to differentiate MS from other mimics. This study aimed to determine the value of CVS as a diagnostic biomarker for distinguishing MS from its mimics. Patients and Methods Patients were prospectively recruited into two groups: a typical clinical (TC) MS presentation with an atypical MRI for MS and an atypical clinical (ATC) MS presentation with a typical MRI for MS. Patients underwent a 1.5T MRI brain scan with a T2*-weighted gradient-echo sequence. The presence of the central vein was assessed by a radiologist blinded to patients’ clinical presentation. The MS consultant made the final diagnosis without reviewing the T2*-weighted gradient-echo sequence or the CVS analysis results. Results Forty-two patients were included. Ten (40%) out of 25 TC patients were diagnosed with clinically definite MS (CDMS), with a mean percentage of CV-positive lesions of 65.5% among CDMS patients. Four (23.5%) out of 17 ATC patients were diagnosed with CDMS with a mean CV-positive lesions percentage of 68.25% among CDMS patients. TC patients who were not diagnosed as CDMS had a mean CV-positive lesions percentage of 10.13%, while ATC patients who were not diagnosed as CDMS had a mean CV-positive lesions percentage of 16.38%. The CVS showed 85.7% sensitivity and 100% specificity (95% confidence interval: 0.919–1.018) for diagnosis of MS at a cut off value of 45% ( p < 0.001). The percentage of CV-positive lesions was significantly higher in oligoclonal bands (OCBs) positive patients compared to OCBs negative patients ( p < 0.001) and those with spinal cord lesions compared to patients with no spinal cord lesions ( p = 0.017). Conclusion The CVS has 85.7% sensitivity and 100% specificity for the diagnosis of MS at a cutoff value of 45%.